scholarly journals Characterization and functional roles of paternal RNAs in 2–4 cell bovine embryos

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nicole Gross ◽  
Maria Giuseppina Strillacci ◽  
Francisco Peñagaricano ◽  
Hasan Khatib
Keyword(s):  
Male Fertility ◽  
Expression Patterns ◽  
Low Fertility ◽  
Regulatory Subunit ◽  
Parental Origin ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Functional Roles ◽  
Cell Embryo

AbstractEmbryos utilize oocyte-donated RNAs until they become capable of producing RNAs through embryonic genome activation (EGA). The sperm’s influence over pre-EGA RNA content of embryos remains unknown. Recent studies have revealed that sperm donate non-genomic components upon fertilization. Thus, sperm may also contribute to RNA presence in pre-EGA embryos. The first objective of this study was to investigate whether male fertility status is associated with the RNAs present in the bovine embryo prior to EGA. A total of 65 RNAs were found to be differentially expressed between 2–4 cell bovine embryos derived from high and low fertility sires. Expression patterns were confirmed for protein phosphatase 1 regulatory subunit 36 (PPP1R36) and ataxin 2 like (ATXN2L) in three new biological replicates. The knockdown of ATXN2L led to a 22.9% increase in blastocyst development. The second objective of this study was to characterize the parental origin of RNAs present in pre-EGA embryos. Results revealed 472 sperm-derived RNAs, 2575 oocyte-derived RNAs, 2675 RNAs derived from both sperm and oocytes, and 663 embryo-exclusive RNAs. This study uncovers an association of male fertility with developmentally impactful RNAs in 2–4 cell embryos. This study also provides an initial characterization of paternally-contributed RNAs to pre-EGA embryos. Furthermore, a subset of 2–4 cell embryo-specific RNAs was identified.

scholarly journals 153EXPRESSION OF TGF-BETA I AND TYPE I AND TYPE II OF TGF-BETA RECEPTORS IN BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
B.K. Kim ◽  
H.J. Chung ◽  
B.C. Yang ◽  
D.H. Kim ◽  
J.H. Woo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Type I ◽  
Bovine Embryo ◽  
Type Ii ◽  
Bovine Embryos ◽  
Mrna And Protein Expression ◽  
Bovine Oocytes

Although the effects of TGFβ1, as an important factor in the mice embryo development have been reported, little information relevant to this subject is known in the bovine embryo. The objectives of this study were to investigate the presence and expression patterns of TGFβ1 and TGFβ1 receptors, types I and II, in unfertilized oocytes and fertilized bovine embryos in normal and NT embryo development. We postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and show different expression patterns at different stages of bovine embryo development. Immature bovine oocytes were aspirated from follicles of ovaries obtained from a local abattoir and they were cultured for up to 24h and fertilized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the presence of TGFβ1 and type I and type II of TGFβ1 receptors (the essential components of the TGFβ1 signaling pathway) in unfertilized oocytes and preimplantation embryos. Also, mRNA and protein expression patterns of TGFβ1 and their receptors at various stages of embryos were examined. It was found that both receptors, as well as TGFβ1, were present in the unfertilized bovine oocytes, indicating that TGFβ1 is a maternally expressed protein. Although the type I TGFβ1 receptor was present at the morulae and blastocyst stages, the type II TGFβ1 receptor was not present at both stages. It was also confirmed that the expression level of TGFβ1 was high at the 8-cell stage, and mRNA and protein expression patterns of TGFβ1 and their receptors were not coincident. Interestingly, TGFβ1 protein was not detected at blastocyst stage of embryos, whereas the mRNA expression level was high at this stage. The results of this experiment indicate that TGFβ1 protein may be needed by embryos after the blastocyst stage and may be expressed in hatched embryos for implantation. These findings support the hypothesis that there may be an interaction between the TGFβ1 and TGFβ1 receptors in the unfertilized oocytes and preimplantation embryos, and that TGFβ1 signaling may be important for the development of the oocytes and the preimplantation embryos.

121 DEVELOPMENTAL CHANGES IN THERMOPROTECTIVE ACTIONS OF INSULIN-LIKE GROWTH FACTOR-1 ON THE PREIMPLANTATION BOVINE EMBRYO

2011 ◽  
Vol 23 (1) ◽  
pp. 165
Author(s):  
A. Q. S. Bonilla ◽  
L. J. Oliveira ◽  
M. Ozawa ◽  
E. M. Newsom ◽  
M. C. Lucy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Heat Shock ◽  
Differentially Expressed Genes ◽  
Blastocyst Stage ◽  
Differentially Expressed ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Developmentally Regulated ◽  
Cell Embryo

Insulin-like growth factor-1 (IGF1) is an important endocrine signal for regulation of early embryonic development. It increases the proportion of preimplantation embryos becoming blastocysts, alters blastocyst gene expression, improves resistance of embryos to various stresses and can enhance survival of embryos after transfer to recipients. The present study had 2 objectives. The first was to determine whether the thermoprotective actions of IGF1 on the preimplantation bovine embryo were developmentally regulated, with the 2-cell embryo being refractory to IGF1. The second was to determine the molecular basis for the improved competence of embryos treated with IGF1 to establish pregnancy after transfer to heat-stressed recipients. Heat shock at 41°C decreased (P < 0.005) the percentage of 2-cell embryos becoming a blastocyst at day 8 (39.5 v. 21% for 38.5 and 41°C, respectively), and treatment of embryos with 100 ng mL–1 IGF1 did not provide thermoprotection to 2-cell embryos heat shocked at 41°C (21 v. 21% for control and IGF1-treated embryos, respectively). Heat shock at 41°C had no effect on blastocyst development of day 5 embryos. However, exposure to 42°C reduced (P < 0.001) blastocyst development of day 5 embryos (87 v. 47.6% for 38.5 and 42°C, respectively). Furthermore, treatment of embryos with 100 ng mL–1 IGF1 reduced (P = 0.05) the effect of heat shock at 42°C on day 5 embryos (48 v. 66% control and IGF1-treated embryos, respectively). Failure of IGF1 to alter 2-cell embryo survival after heat shock was not associated with reduced expression of genes involved in IGF1 signaling (IGF1R, RAF1, PI3K, and MAPK), as shown by quantitative real-time RT-PCR assay, or in amounts of immunoreactive IGF1R protein. Treatment with IGF1 had little effect on the transcriptome at the blastocyst stage, with a total of 102 differentially expressed genes identified. Among the differentially expressed genes were several involved in apoptosis, protection against free radicals, and development. Changes in gene expression are consistent with IGF1 acting to induce an anti-apoptotic state and inhibit neurulation. In conclusion, thermoprotective actions of IGF1 are developmentally regulated. Failure of IGF1 to protect the 2-cell embryo from heat shock could reflect the fact that these embryos are maximally sensitive to damage caused by heat shock or reflect the quiescence of the embryonic genome at this early stage in development. Changes in gene expression at the blastocyst stage induced by IGF1 could contribute to the increased survival of IGF1-treated embryos when transferred during periods of heat stress. Support: USDA NRI 2007-35203-18070 and 2009-65203-05732.

scholarly journals Improvement of in vitro-produced bovine embryo treated with coagulansin-A under heat-stressed condition

Reproduction ◽  
2017 ◽  
Vol 153 (4) ◽  
pp. 421-431 ◽  
Author(s):  
Imran Khan ◽  
Kyeong-Lim Lee ◽  
Lianguang Xu ◽  
Ayman Mesalam ◽  
M M R Chowdhury ◽  
...  
Keyword(s):  
Heat Stress ◽  
Expression Patterns ◽  
Treated Group ◽  
Cell Number ◽  
Control Group ◽  
Bovine Embryo ◽  
Stressed Condition ◽  
Blastocyst Development ◽  
Different Temperatures

Heat stress has large effects on reproduction including conception rate in cattle. In this study, we examined the effects of coagulansin-A (coa-A), a steroidal lactone, on acquired thermo tolerance duringin vitroproduction of bovine embryos. Oocytes were incubated inin vitromaturation (IVM) media with or without coa-A at two different temperatures, 40.5˚C and 42˚C, for 20 h. The treatment of coa-A significantly improved blastocyst development only at 40.5˚C (P < 0.05). Interestingly, immunofluorescence analysis demonstrated that coa-A induced heat shock protein 70 (HSP70) and phosphatidylinositol-3-kinase (PI3K), but significantly attenuated nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX2). To determine the expression patterns of related genes at the transcription level, qRT-PCR was performed. Expression ofHSP70andPI3Kwas elevated, whereas expression ofNF-κB,COX2and inducible nitric oxide synthase (iNOS) was significantly (P < 0.05) downregulated in the coa-A-treated group compared with the control group. Moreover, pro-apoptotic genes were downregulated, and antiapoptic genes were upregulated in the coa-A group. We also counted the total cell number and apoptotic nuclei at the blastocyst and found that more cell numbers (143.1 ± 1.5) and less apoptotic damages (6.4 ± 0.5) in the coa-A treatment group comparing to control group (131.4 ± 2.0 and 10.8 ± 0.5), indicating the enhanced embryo quality. In conclusion, our results demonstrate that the coa-A not only improved the blastocyst developmentin vitrobut also increased their resistance to heat stress condition through induction ofHSP70/PI3K.

Effect of α-tocopherol on in vitro culture of bovine embryos

2007 ◽  
Vol 87 (4) ◽  
pp. 539-542 ◽  
Author(s):  
A. Marques ◽  
G. Antunes ◽  
P. Santos ◽  
A. Chaveiro ◽  
F. Moreira da Silva
Keyword(s):  
Culture Media ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Media Control ◽  
Oviductal Fluid ◽  
Bovine Oocytes ◽  
Key Words Antioxidant

Bovine oocytes were matured and fertilised under 5% CO2. Presumptive zygotes were co-cultured in synthetic oviductal fluid droplets, supplemented with either 0 (control), 25, 50, 100, or 200 µM of α-tocopherol. Blastocyst development rates were significantly influenced (P < 0.05) by the level of antioxidant in the culture media. Control showed a blastocyst yield of 18.46, 21.11, 27.92, and 31.66%, respectively, at α25; α50 and α100. Blastocyst yield for α200 was severely decreased, to 8.01%. An increase in overall IVF results was also observed as the concentration of α-tocopherol increased (control, 14.21%; α25, 14.35%; α50, 19.52%; α100, 21.11%), decreasing to 5.75% for the concentration of α200. The present study clearly demonstrates that α-tocopherol at a concentration of 100 µM significantly improves the proportion of oocytes that develop to the blastocyst stage. Key words: Antioxidant, α-tocopherol, reactive oxygen species, bovine, embryo

Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development

2005 ◽  
Vol 17 (2) ◽  
pp. 23 ◽  
Author(s):  
Christine Wrenzycki ◽  
Doris Herrmann ◽  
Andrea Lucas-Hahn ◽  
Karin Korsawe ◽  
Erika Lemme ◽  
...  
Keyword(s):  
Messenger Rna ◽  
De Novo ◽  
Culture Media ◽  
Expression Patterns ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Developmental Abnormalities ◽  
Embryonic Genome

The preimplantation bovine embryo is initially under the control of maternal genomic information that is accumulated during oogenesis. The genetic programme of development soon becomes dependent on new transcripts derived from activation of the embryonic genome. The early steps in development, including the timing of the first cleavage, activation of the embryonic genome, compaction and blastocyst formation, can be affected by the culture media and conditions, as well as the production procedure itself. These perturbations can possibly result in a marked decrease in the quality of the resulting blastocysts and may even affect the viability of offspring born after transfer. In vitro procedures such as in vitro production and somatic nuclear transfer of bovine embryos have been shown to be correlated with significant up- or downregulation, de novo induction or silencing of genes critical for undisturbed fetal and neonatal development. These alterations are likely to be caused by epigenetic modifications, such as DNA methylation and histone modifications. Analysis of perturbed epigenetic reprogramming and of the related phenomena, such as genomic imprinting and X-chromosome inactivation, in bovine embryos is promising for understanding the underlying mechanisms of developmental abnormalities, such as large offspring syndrome.

scholarly journals Individual commitment to a group effect: strengths and weaknesses of bovine embryo group culture

Reproduction ◽  
2014 ◽  
Vol 148 (5) ◽  
pp. 519-529 ◽  
Author(s):  
Eline Wydooghe ◽  
Leen Vandaele ◽  
Sofie Piepers ◽  
Jeroen Dewulf ◽  
Etienne Van den Abbeel ◽  
...  
Keyword(s):  
Classical Group ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Group Culture ◽  
Individual Culture ◽  
Better Than

Recently, new culture devices such as Corral and Primo Vision dishes have been designed for the culture of human embryos to allow the combination of group culture plus follow-up of individual embryos. Bovine inseminated oocytes were allocated to Primo Vision dishes, Corral dishes, individual culture or classical group culture. Blastocyst development in Primo Vision dishes was similar to classical group culture (34.3 and 39.0% respectively), and better than Corral dishes or individual culture (28.9 and 28.5% respectively). In Primo Vision dishes, a higher number of ‘slow’ embryos developed to the blastocyst stage compared with their individually cultured counterparts, while no differences were observed for ‘fast’ embryos. ‘Slow’ embryos in a ‘standard drop’ had a higher chance of becoming a blastocyst compared with individual culture (OR: 2.3), whereas blastulation of ‘fast’ embryos was less efficient in a ‘delayed drop’ than in individual culture (OR: 0.3). The number of non-cleaved embryos in Primo Vision dishes did not negatively influence blastocyst development. Likewise, removing non-cleaved embryos (NC removed) and regrouping the cleaved embryos afterwards (ReGR) did not affect blastocyst development and quality compared with group culture in Primo Vision dishes (CTRL, 31.6%, NC removed, 29.3% and ReGR, 29.6%). The experiments revealed that group culture of bovine embryos in Primo Vision dishes is superior to individual culture, primarily because of the higher blastocyst rate achieved by slow embryos. Non-cleaved or arrested embryos do not hamper the ability of co-cultured bovine embryos to reach the blastocyst stage in group culture.

Fine-structural cytochemical and immunocytochemical observations on nuclear bodies in the bovine 2-cell embryo

Zygote ◽  
2000 ◽  
Vol 8 (4) ◽  
pp. 315-328 ◽  
Author(s):  
V. Kopecný ◽  
M. Biggiogera ◽  
J. Pivko ◽  
A. Pavlok ◽  
T.E. Martin ◽  
...  
Keyword(s):  
Nuclear Bodies ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Cytological Evidence ◽  
Somatic Cell Nucleus ◽  
Cell Embryo ◽  
Sm Antigen

Nuclear bodies occuring during the 2-cell stage of bovine embryos (obtained either by in vitro fertilisation of in vitro matured ovarian oocytes, or collection after fertilisation and cleavage in vivo) were studied using ultrastructural cytochemistry and immunocytochemistry to determine whether their occurrence may be linked with the onset of embryonic transcription. In addition, the species-specific ultrastructural features of the interchromatin structures of the 2-cell bovine embryo were displayed. Three different types of nuclear bodies were distinguished: (i) nucleolus precursor bodies (NPBs), (ii) loose bodies (LBs) and (iii) dense bodies (DBs). In order to determine their possible functional significance, we considered parallels between these three nuclear entities and interchromatin compartments reported in other cells. As detected by their preferential ribonucleoprotein staining, all types of nuclear bodies contained ribonucleoproteins. In contrast to the other types of nuclear bodies studied, NPBs contained argyrophilic proteins but in no case they did show morphological features of functional nucleoli. Both compact and vacuolated forms of NPBs were seen in both in vivo and in vitro embryos, sometimes simultaneously in the same nucleus. LBs and DBs reacted with antibodies to Sm antigen, indicating the presence of a group of nucleoplasmic, non-nucleolar small nuclear ribonucleoproteins (snRNPs). The immunoreactivity for Sm antigen was more intense and homogeneous in DBs than in LBs. DBs were seen in both categories of embryo. A possible kinship of DBs with the sphere organelle known from oocytes of different animal species or the prominent spherical inclusions of the early mouse embryo nuclei is suggested. The last type of intranuclear body, the LBs, showed a composite structure. Their granular component, occurring in clusters and displaying immunoreactivity for Sm antigen, was similar to interchromatin granules and was therefore named IG-like granules. Another component forming the LBs showed a much finer structure and a lower immunoreactivity with anti-Sm antibodies. We suggest that this amorphous component may be related to the IG-associated zone. All three types of intranuclear bodies were often seen close together, suggesting their possible mutual functional relationship. From these and other observations we conclude that the intranuclear bodies in 2-cell bovine embryos correspond, with the exception of the NPB, to similar structures/compartments supposed to accumulate inactive spliceosomal components in certain phases of somatic cell nucleus functions. Accordingly, the occurrence of such nuclear bodies does not represent cytological evidence for RNA synthesis. In contrast to this, an important morphological feature revealing the status of the bovine 2-cell embryo is the vacuol-isation of the NPB.

scholarly journals Evaluation of Bovine Embryo Biopsy Techniques according to Their Ability to Preserve Embryo Viability

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
M. Cenariu ◽  
E. Pall ◽  
C. Cernea ◽  
I. Groza
Keyword(s):  
Pregnancy Rate ◽  
Genetic Diagnosis ◽  
Needle Aspiration ◽  
Embryo Biopsy ◽  
Embryo Cryopreservation ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Biopsy Methods ◽  
Biopsy Method

The purpose of this research was to evaluate three embryo biopsy techniques used for preimplantation genetic diagnosis (PGD) in cattle and to recommend the least invasive one for current use, especially when PGD is followed by embryo cryopreservation. Three hundred bovine embryos were biopsied by either one of the needle, aspiration or microblade method, and then checked for viability by freezing/thawing and transplantation to recipient cows. The number of pregnancies obtained after the transfer of biopsied frozen/thawed embryos was assessed 30 days later using ultrasounds. The results were significantly different between the three biopsy methods: the pregnancy rate was of 57% in cows that received embryos biopsied by needle, 43% in cows that received embryos biopsied by aspiration, and 31% in cows that received embryos biopsied by microblade. Choosing an adequate biopsy method is therefore of great importance in embryos that will undergo subsequent cryopreservation, as it significantly influences their viability after thawing.

scholarly journals SOS2 Comes to the Fore: Differential Functionalities in Physiology and Pathology

2021 ◽  
Vol 22 (12) ◽  
pp. 6613
Author(s):  
Fernando C. Baltanás ◽  
Rósula García-Navas ◽  
Eugenio Santos
Keyword(s):  
Expression Patterns ◽  
Critical Role ◽  
Ras Signaling ◽  
Epidermal Stem Cell ◽  
Functional Specificity ◽  
Functional Roles ◽  
Signaling Axis ◽  
Therapy Target ◽  
External Stimuli

The SOS family of Ras-GEFs encompasses two highly homologous and widely expressed members, SOS1 and SOS2. Despite their similar structures and expression patterns, early studies of constitutive KO mice showing that SOS1-KO mutants were embryonic lethal while SOS2-KO mice were viable led to initially viewing SOS1 as the main Ras-GEF linking external stimuli to downstream RAS signaling, while obviating the functional significance of SOS2. Subsequently, different genetic and/or pharmacological ablation tools defined more precisely the functional specificity/redundancy of the SOS1/2 GEFs. Interestingly, the defective phenotypes observed in concomitantly ablated SOS1/2-DKO contexts are frequently much stronger than in single SOS1-KO scenarios and undetectable in single SOS2-KO cells, demonstrating functional redundancy between them and suggesting an ancillary role of SOS2 in the absence of SOS1. Preferential SOS1 role was also demonstrated in different RASopathies and tumors. Conversely, specific SOS2 functions, including a critical role in regulation of the RAS–PI3K/AKT signaling axis in keratinocytes and KRAS-driven tumor lines or in control of epidermal stem cell homeostasis, were also reported. Specific SOS2 mutations were also identified in some RASopathies and cancer forms. The relevance/specificity of the newly uncovered functional roles suggests that SOS2 should join SOS1 for consideration as a relevant biomarker/therapy target.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

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